Corneal hydration is derived almost exclusively from the ion transport activities of the corneal endothelium. Numerous studies have shown that endothelial fluid transport, dehydration activity, and electrical potential are dependent on bathing solution pH and the presence of bicarbonate. In an environment of constant carbon dioxide concentration, extracellular and intracellular bicarbonate concentrations are exquisitely determined by extra- and intracellular pH (pHo and pHi), respectively. This suggest that elucidation of pHi regulatory mechanisms will be necessary to form any meaningful model for endothelial fluid transport. Preliminary observations have shown that endothelial pHi can be measured in fresh corneal explants by use of pH sensitive intracellular fluorescent probes. This one-year pilot project proposes to use this technique to: (1) identify the pHi regulatory mechanisms in fresh bovine corneal endothelium; (2) determine the relative activities of the bicarbonate transporters; and (3) determine the apical-basal polarity of the transporters by establishing cultures of bovine endothelium with the intent of growing monolayers onto tissue culture filters for independent perfusion of apical and basal membranes. Completion of these aims will provide data to formulate a working model of bicarbonate transport across a mammalian corneal endothelium. The long range goals of this line of study will be to: 1) correlate bicarbonate transporter activities with fluid transport and/or electrical potential; 2) measure bicarbonate transport activities in post-surgical diseased human endothelium to ascertain their role in the decline of endothelial function; and 3) determine the effects of contact lens induced acidosis on endothelial bicarbonate transport.